The goal of this proposal is the discovery and development of novel drugs to be used in a disease intervention strategy for influenza (flu) infection that comprise short interfering RNAs (siRNAs), the molecules that induce RNA interference. To achieve this goal we will design, synthesize and test a pool of chemically optimized siRNAs targeting highly conserved flu gene sequences by in vitro and in vivo screens against low and highly pathogenic flu strains, including H5N1. Importantly, our siRNA intervention strategy for flu infection will investigate the incorporation of both stabilization chemistries and novel "universal base" chemistry: Stabilization chemistries are designed to confer additional drug-like properties on our siRNAs and yield molecules with significantly increased half-lives in vitro and in vivo. Universal bases are able to pair with all nucleotides and can be incorporated at any position within a siRNAs to overcome the challenges of designing anti-viral siRNAs due to sequence variability across multiple viral strains or different viral species. For this Phase I SBIR, our goal is to apply these novel approaches toward pulmonary control of flu infections where we already have vast experience developing RNAi therapeutics. With novel chemistry, we aim to achieve a siRNA therapeutic that is active across a broad range of flu strains and applicable for both prophylaxis and treatment. The specific aims of this research proposal are: 1) Bioinformatic identification of conserved viral gene targets: Use sequence alignments and other algorithms to identify siRNA targets across flu strains, including the avian flu strain H5N1; 2) siRNA design and synthesis: Design and synthesize siRNA molecules to the conserved viral gene targets identified in specific aim #1 incorporating stabilization chemistry at appropriate positions and universal bases at sites complimentary to hypervariable sites; 3) Specific Aim 3-In vitro efficacy and stability screen: Screen siRNAs from specific aim #2 for anti-viral activity against low and highly pathogenic flu strains, as well as for stability-the most potent and stable siRNAs will be designated lead candidates; 4) Efficacy testing in animals: Test lead candidate siRNAs from specific aim #3 for efficacy in mouse (prophylaxis and treatment) infected with low and highly pathogenic flu strains. At the end of this Phase I research, we will have identified highly potent siRNA(s) that will be further evaluated in other in vivo models that include ferrets. Ultimately, efficacious candidates will be selected for safety testing in ore-clinical pharmacology and GLP toxicology studies. [unreadable] [unreadable] [unreadable]